title: A guanosine 3',5'-cyclic monophosphate (cGMP) reporter system based on the G-kinase/CREB/CRE signal transduction pathway.
abstract: Guanylate cyclases constitute a gene family of enzymes that synthesize the second messenger guanosine 3',5'-cyclic monophosphate (cGMP) and play important roles in diverse physiological functions. Here we report a novel, simple and highly sensitive method for measurement intracellular cGMP concentrations using a cAMP-responsive element (CRE) and cGMP-dependent protein kinase (cGK). Transient transfection of the CRE reporter plasmid, encoding a luciferase reporter gene under the control of a modified promoter containing a CRE, and a cGK expression vector into HEK293 cells followed by treatment with 8-bromo-cGMP showed a dose dependent increase in luciferase activity. Moreover, HEK293 cells expressing GC-A or GC-B natriuretic peptide receptors and harboring this reporter system responded to specific ligands in a dose dependent manner. Our results indicate that this reporter gene method enables high throughput screening of receptor-type GC selective agonists in the treatment of cardiovascular diseases and homeostatic dysfunctions.
authors.affiliation: Department of Biochemistry, National Cerebral and Cardiovascular Center Research Institute, Fujishiro-dai, Suita, Osaka, Japan. okano@ri.ncvc.go.jp
authors.forename: Ichiro
authors.initials: I
authors.lastname: Okano
authors.forename: Mikiya
authors.initials: M
authors.lastname: Miyazato
authors.forename: Kenji
authors.initials: K
authors.lastname: Kangawa
mesh_terms: D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism*; D006152:Cyclic GMP / Q000031:analogs & derivatives / Q000032:analysis* / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology; D062965:Cyclic GMP-Dependent Protein Kinase Type I; D017869:Cyclic GMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics; D004353:Drug Evaluation, Preclinical; D017930:Genes, Reporter / Q000187:drug effects; D057809:HEK293 Cells; D006801:Humans; D050705:Receptors, Guanylate Cyclase-Coupled / Q000819:agonists*; D020218:Response Elements; D015398:Signal Transduction
chemical_list: D017362:Cyclic AMP Response Element-Binding Protein; C016276:8-bromocyclic GMP; D062965:Cyclic GMP-Dependent Protein Kinase Type I; D017869:Cyclic GMP-Dependent Protein Kinases; C569718:PRKG1 protein, human; D050705:Receptors, Guanylate Cyclase-Coupled; D006152:Cyclic GMP